Screening, Identification Of Receptor Protein Of BmCPV And Molecular Mechanism Of Wing Disc Development In Silkworm, Bombyx Mori

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) can infect silkworm mid gutcells, leading to the occurrence of the most harmful disease in sericulture-midgutpolyhedrosis. In order to identify the receptor protein of BmCPV and explore themolecular mechanism of BmCPV invading silkworm cells, this study screened midgutcells (membrane) protein using three frequently used screening methods such as Co-IP,VOPBA, pull-down etc., as well as screening method of protein-protein interaction inPVDF membrane with the anti-BmCPV virion antibody, anti-structural protein VP7antibody and anti-S1encoded sORF of virus genome antibody. Combined withbioinformatics analysis by mass spectrometry,10kinds of candidate receptor proteinswere initially screened: Gcy, AP, BMKT, CRT, CLAT, IPOA, ITGB, BmSRC, RACKand OBP.BmCPV specifically infected silkworm midgut, and tissue-specific expression of thereceptor gene may be associated with tissue-specific infection of virus. Therefore theexpressions of10kinds of candidate receptor gene in various tissues of fifth instarsilkworm were investigated. The results show that: the selected10genes were allexpressed in various tissues, there are large differences in gene expression levels invarious tissues, and the expression levels of different candidate receptor genes are notthe same in the same organization. Opioid-binding protein genes expressed at highlevels in midgut. We speculated that the tissue specificity of BmCPV infecting is notonly impacted by tissue-specificity of receptor, but also by the role of some otherfactors.To further identify the function of candidate receptor gene of BmCPV, this study designed and synthesized three kinds of sequence-specific siRNA according to thesequence of each gene, and investigated the effect of silencing candidate receptor geneon BmCPV level in cultured BmN cells and silkworm individual. The results showedthat: the relative expression levels of BmCPV S1were down-regulated more than90%compared to the control group after silencing AP, CRT, CLAT, IPOA and RACK gene bytransfecting siRNA in BmN cells; and the relative expression levels of VP1weredown-regulated more than90%compared to the control group after inhibiting BMKT,CLAT, TPOA, ITGB and BmSRC gene expression by injecting siRNA at48h afterinfecting BmCPV. This indicated that the encoded product of above-described genesmay be members of BmCPV receptor complex, playing an important role in theinvasion process of BmCPV.In order to further identify candidate receptor protein, prokaryotic expressionrecombinant vector of AP, CLAT, ITGB and RACK were constructed, and the polyclonalantibody were prepared using inducible expressed recombinant proteins. The results ofantibody blocking experiments showed that compared to the control group, both100μg/mL and300μg/mL concentrations of anti-candidate receptor antibody couldsignificantly reduced relative expression level of VP1gene in BmN cells. These resultsfurther confirmed the possibility of the four candidate receptor as members of BmCPVreceptor complex.Wing disc is mode organ in studying development of insect organs. In order toinvestigate the regulation mechanism of the organ development in silkworm, this studycarried out the study on molecular regulation of wing disc development by the methodof comparative proteomics and comparative transcriptomics. Proteomic profles fromthe wing discs of silkworms at the larval, pupal, and adult moth stages were determinedusing shotgun proteomics and MS sequencing. We identifed241,218, and223proteinsfrom the larval, pupal, and adult moth stages, respectively, of which139were shared byall three stages. In addition, there were55,37, and43specifc proteins identifed at thelarval, pupal, and adult moth stages, respectively. Gene ontology (GO) analysis ofproteins specifc to each of these three stages enabled their association to cellular component, molecular function, and biological process categories. The analysis ofsimilarities and differences in these identified proteins will greatly further ourunderstanding of wing disc development in silkworm.To further understand the molecular mechanisms of wing disc growth anddevelopment in silkworm, RNA-Seq were performed on the wing disc of larvae (fifthday of the fifth instar) and pupa (seventh day of the pupal stage), and the wing of moth(first day of the moth stage). Clean data in three different developmental stages of theraw data after removal of low value sequences were35973346,41374988and37243758reads, the average length of each reads was89.25bp, and the effective rate ofdata was72.14%. The number of genes in three different developmental stages was11563,11414and11642respectively. GO enrichment (P≤0.05) analysis result showedthat compared with the larval stage, the up (and down) expressed genes of pupal andmoth stages mainly enriched in229(220) and251(225) GO terms and compared withthe pupal stage, the up (and down) expressed genes of moth stage were mainly enrichedin238(198) GO terms. KEGG enrichment analysis result of differentially expressedgenes (P≤0.05) showed that compared with the wing discs of larval stage, the up (anddown) expressed genes of pupal and moth stages were mainly enriched in72(34) and94(35) KEGG pathways (P≤0.05); compared with the pupal stage, the up (and down)expressed genes of moth stages were mainly enriched in the113(24) KEGG pathways.9genes were randomly selected to detect the relative expression levels in wingdiscs at larva, pupa and adult stages by qRT-PCR. The results showed that there wereslight differences between the results of RNA-seq and qRT-PCR, but the overall trend isconsistent, indicating the results of high-throughput sequencing were credible. Theresults provided new clues for further revealing the molecular mechanism of silkwormwing discs growth and development.